Insight into posttranscriptional regulation and global regulatory circuitry will be sought through the study of the Csr system of Escherichia coli. Csr includes: CsrA, an RNA binding protein that regulates translation and/or mRNA stability of numerous target mRNAs; CsrB and CsrC, sRNAs that sequester and antagonize CsrA; BarA-UvrY, a two-component signal transduction system that activates csrB and csrC transcription; and CsrD, a protein that forms a complex with EIIAGlc, activating it to target CsrB/C RNAs for degradation by RNase E. CsrA regulates metabolism, motility and multicellular behavior on a multi-systems scale, as well as virulence circuits of numerous pathogens. Our transcriptomics studies identified hundreds of RNAs that bind to CsrA in vivo. Csr is reciprocally linked to a number of other global regulatory circuits. This complex circuitry allows Csr to reinforce the regulatory effects of multiple stress response systems at a posttranscriptional level. The specific aims are: 1) Footprinting of the CsrA-RNA transcriptome. We are developing next-gen sequencing methods to derive in vivo protein binding and secondary structure information for the transcriptome. We will apply this innovative approach to identify CsrA-RNA footprints and RNA structural changes caused by CsrA binding throughout the transcriptome. The results will suggest new regulatory mechanisms and roles for CsrA and will have a broad and powerful impact on the field of RNA biology. 2) Regulation of sRNA-mRNA basepairing by CsrA. Transcriptomics and gel shift studies show direct CsrA binding to several basepairing sRNAs and suggest that CsrA-sRNA binding influences sRNA-mRNA pairing and regulation. Genetic and biochemical studies will be used to investigate CsrA interaction with a model sRNA and its effects on sRNA structure, Hfq-sRNA binding, and other features of sRNA-mRNA regulation. 3) The critical role(s) of CsrA in bacterial growth. A csrA deletion (?csrA) causes severe growth defects, however the way in which CsrA supports bacterial growth is poorly understood. Using an ?evolve and resequence? approach, we monitored the rapid adaptive evolution of several independent ?csrA populations. Potential ?csrA suppressors were sequenced in genes with known regulatory connections to CsrA and with no known relationship. Single and multiple mutant combinations will be reconstructed using CRISPR-Cas9 and tested for effects on ?csrA growth to authenticate suppressors. Pathways for suppression and the inferred roles of CsrA in supporting growth will be confirmed using transcriptomics and other analyses, thereby uncovering crucial functions of CsrA for growth. Our long-range objectives are to fully understand the components, molecular mechanisms, genetic circuitry and biological functions of Csr, thereby defining the principles that underpin a regulatory super-network. Csr (Rsm) controls the expression of virulence factors, transmission traits and/or persistence of numerous pathogens. Thus, our fundamental discoveries will continue to provide a framework for researchers studying the Csr system in bacterial pathogens, and may inform the use of CsrA as a therapeutic target.